Monalisa Sampaio Carneiro

Sugarcane improvement: how far can we go?

In recent years, efforts to improve sugarcane have focused on the development of biotechnology for this crop. It has become clear that sugarcane lacks tools for the biotechnological route of improvement and that the initial efforts in sequencing ESTs had limited impact for breeding. Until recently, the models used by breeders in statistical genetics approaches have been developed for diploid organisms, which are not ideal for a polyploid genome such as that of sugarcane. Breeding programs are dealing with decreasing yield gains. The contribution of multiple alleles to complex traits such as yield is a basic question underlining the breeding efforts that could only be addressed by the development of specific tools for this grass. However, functional genomics has progressed and gene expression profiling is leading to the definition of gene networks. The sequencing of the sugarcane genome, which is underway, will greatly contribute to numerous aspects of research on grasses. We expect that both the transgenic and the marker-assisted route for sugarcane improvement will contribute to increased sugar, stress tolerance, and higher yield and that the industry for years to come will be able to rely on sugarcane as the most productive energy crop.

Building the sugarcane genome for biotechnology and identifying evolutionary trends.

BackgroundSugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome.ResultsThree hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences.ConclusionThis release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery.

SNP genotyping allows an in-depth characterisation of the genome of sugarcane and other complex autopolyploids

Many plant species of great economic value (e.g., potato, wheat, cotton, and sugarcane) are polyploids. Despite the essential roles of autopolyploid plants in human activities, our genetic understanding of these species is still poor. Recent progress in instrumentation and biochemical manipulation has led to the accumulation of an incredible amount of genomic data. In this study, we demonstrate for the first time a successful genetic analysis in a highly polyploid genome (sugarcane) by the quantitative analysis of single-nucleotide polymorphism (SNP) allelic dosage and the application of a new data analysis framework. This study provides a better understanding of autopolyploid genomic structure and is a sound basis for genetic studies. The proposed methods can be employed to analyse the genome of any autopolyploid and will permit the future development of high-quality genetic maps to assist in the assembly of reference genome sequences for polyploid species.

De Novo Assembly and Transcriptome Analysis of Contrasting Sugarcane Varieties

Sugarcane is an important crop and a major source of sugar and alcohol. In this study, we performed de novo assembly and transcriptome annotation for six sugarcane genotypes involved in bi-parental crosses. The de novo assembly of the sugarcane transcriptome was performed using short reads generated using the Illumina RNA-Seq platform. We produced more than 400 million reads, which were assembled into 72,269 unigenes. Based on a similarity search, the unigenes showed significant similarity to more than 28,788 sorghum proteins, including a set of 5,272 unigenes that are not present in the public sugarcane EST databases; many of these unigenes are likely putative undescribed sugarcane genes. From this collection of unigenes, a large number of molecular markers were identified, including 5,106 simple sequence repeats (SSRs) and 708,125 single-nucleotide polymorphisms (SNPs). This new dataset will be a useful resource for future genetic and genomic studies in this species.

The characterization of a new set of EST-derived simple sequence repeat (SSR) markers as a resource for the genetic analysis of Phaseolus vulgaris

BackgroundOver recent years, a growing effort has been made to develop microsatellite markers for the genomic analysis of the common bean (Phaseolus vulgaris) to broaden the knowledge of the molecular genetic basis of this species. The availability of large sets of expressed sequence tags (ESTs) in public databases has given rise to an expedient approach for the identification of SSRs (Simple Sequence Repeats), specifically EST-derived SSRs. In the present work, a battery of new microsatellite markers was obtained from a search of the Phaseolus vulgaris EST database. The diversity, degree of transferability and polymorphism of these markers were tested.ResultsFrom 9,583 valid ESTs, 4,764 had microsatellite motifs, from which 377 were used to design primers, and 302 (80.11%) showed good amplification quality. To analyze transferability, a group of 167 SSRs were tested, and the results showed that they were 82% transferable across at least one species. The highest amplification rates were observed between the species from the Phaseolus (63.7%), Vigna (25.9%), Glycine (19.8%), Medicago (10.2%), Dipterix (6%) and Arachis (1.8%) genera. The average PIC (Polymorphism Information Content) varied from 0.53 for genomic SSRs to 0.47 for EST-SSRs, and the average number of alleles per locus was 4 and 3, respectively. Among the 315 newly tested SSRs in the BJ (BAT93 X Jalo EEP558) population, 24% (76) were polymorphic. The integration of these segregant loci into a framework map composed of 123 previously obtained SSR markers yielded a total of 199 segregant loci, of which 182 (91.5%) were mapped to 14 linkage groups, resulting in a map length of 1,157 cM.ConclusionsA total of 302 newly developed EST-SSR markers, showing good amplification quality, are available for the genetic analysis of Phaseolus vulgaris. These markers showed satisfactory rates of transferability, especially between species that have great economic and genomic values. Their diversity was comparable to genomic SSRs, and they were incorporated in the common bean reference genetic map, which constitutes an important contribution to and advance in Phaseolus vulgaris genomic research.

GBS-based single dosage markers for linkage and QTL mapping allow gene mining for yield-related traits in sugarcane

BackgroundSugarcane (Saccharum spp.) is predominantly an autopolyploid plant with a variable ploidy level, frequent aneuploidy and a large genome that hampers investigation of its organization. Genetic architecture studies are important for identifying genomic regions associated with traits of interest. However, due to the genetic complexity of sugarcane, the practical applications of genomic tools have been notably delayed in this crop, in contrast to other crops that have already advanced to marker-assisted selection (MAS) and genomic selection. High-throughput next-generation sequencing (NGS) technologies have opened new opportunities for discovering molecular markers, especially single nucleotide polymorphisms (SNPs) and insertion-deletion (indels), at the genome-wide level. The objectives of this study were to (i) establish a pipeline for identifying variants from genotyping-by-sequencing (GBS) data in sugarcane, (ii) construct an integrated genetic map with GBS-based markers plus target region amplification polymorphisms and microsatellites, (iii) detect QTLs related to yield component traits, and (iv) perform annotation of the sequences that originated the associated markers with mapped QTLs to search putative candidate genes.ResultsWe used four pseudo-references to align the GBS reads. Depending on the reference, from 3,433 to 15,906 high-quality markers were discovered, and half of them segregated as single-dose markers (SDMs) on average. In addition to 7,049 non-redundant SDMs from GBS, 629 gel-based markers were used in a subsequent linkage analysis. Of 7,678 SDMs, 993 were mapped. These markers were distributed throughout 223 linkage groups, which were clustered in 18 homo(eo)logous groups (HGs), with a cumulative map length of 3,682.04 cM and an average marker density of 3.70 cM. We performed QTL mapping of four traits and found seven QTLs. Our results suggest the presence of a stable QTL across locations. Furthermore, QTLs to soluble solid content (BRIX) and fiber content (FIB) traits had markers linked to putative candidate genes.ConclusionsThis study is the first to report the use of GBS for large-scale variant discovery and genotyping of a mapping population in sugarcane, providing several insights regarding the use of NGS data in a polyploid, non-model species. The use of GBS generated a large number of markers and still enabled ploidy and allelic dosage estimation. Moreover, we were able to identify seven QTLs, two of which had great potential for validation and future use for molecular breeding in sugarcane.

Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane

Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

Genetic control of Fusarium wilt in common bean

O objetivo do trabalho foi determinar o controle genetico da resistencia do feijoeiro comum ao patotipo FOP 46 de Fusarium oxysporum f. sp. phaseoli e estimar parâmetros geneticos da resistencia a doenca. Em casa de vegetacao, as avaliacoes da resistencia foram conduzidas em familias F2:3 dos cruzamentos Milionario 1732 x Macanudo e FT-Taruma x Macanudo. Os sintomas da doenca foram avaliadas nas plantas aos doze dias apos a inoculacao, atribuindo-se notas que variavam de 1 (plantas sem sintomas) a 9 (plantas totalmente murchas e ou mortas). A analise das frequencias observadas de plantas resistentes e suscetiveis na populacao F2:3 resultantes da autofecundacao das plantas F2 dos cruzamentos Milionario 1732 x Macanudo e FT Taruma x Macanudo, sugeriu um gene efeito maior com acao genica de dominância incompleta. O aparecimento na familia F2:3 de genotipos transgressivos sugere a natureza oligogenica da resistencia da murcha do fusario. As estimativas de herdabilidade e predicao de ganho por selecao foram elevadas em ambos os cruzamentos.

Using quantitative PCR with retrotransposon-based insertion polymorphisms as markers in sugarcane

Highlight qPCR-RBIP was used to examine the dosage of particular markers and evolutionary history in Saccharum and the related genera, Erianthus and Miscanthus. It also differentiated between S. spontaneum and S. officinarum.

Breeding of Sugarcane

Sugarcane is the main source for sugar production and the most important crop for energy production, as well as for byproducts like ethanol and fibers in the world. With a complex genome, the plant has its species from crosses between species of the genus Saccharum, which were the basis for sugarcane breeding programs worldwide. The production of sugarcane has increased worldwide due to breeding programs that have developed more productive clones for specific uses and adapted to different climatic conditions. The future objective of breeding programs is to develop sugarcane with high productivity, high sucrose content, drought tolerance, and high production of ethanol and biomass, i.e., plants with high fiber content and with cell walls easily broken to favor the production of ethanol from bagasse, efficient plants with low nitrogen fertilizer use, and others, and consequently to reduce environmental impacts. Currently, the demand for products derived from sugarcane is consistently increasing; the ethanol byproduct has been pointed out as one of the important sources to feed the demand for renewable energy in fossil and nonrenewable fuel substitution programs in different countries around the world. This chapter describes the genetic improvement of sugarcane and its current goals.